The present invention relates to sulfur- and selenium-containing compounds and methods for using these compounds to protect mammals from toxic insults. More specifically, the present invention relates to prodrugs and conjugates of thiol- or selenol-containing compounds, such as cysteine, cysteamine, glutathione, selenocysteine, selenocysteamine, and the Walter Reed (WR) compounds.
Technical Background
Thiol- or selenol-containing compounds, e.g., cysteine, cysteamine, glutathione, selenocysteine, selenocysteamine, and the WR compounds, are known protective and preventive agents. Potential protective or preventive uses of such agents are widespread, as in reducing the unwanted side effects of chemo- or radiotherapy of cancer, improving cardiovascular function, preventing mutagenesis, preventing the initiation and/or progression of cancer, reducing toxic consequences of planned or unplanned radiation or chemical exposures, slowing the aging process, and preventing cataract formation. New evidence also links these compounds to altered gene expression and enhanced cellular repair processes.
The activity of these thiol- or selenol-containing compounds is mainly due to the sulfur or selenium atom participating in nucleophilic attack on toxic electrophiles, scavenging free radicals, effecting repair of damaged targets through hydrogen atom donation, altering the redox status of the cell, or affecting gene transcription or protein function.
For example, the reduced form of glutathione (Glu-Cys-Gly), a naturally occurring tripeptide with a free sulfhydryl group (SH), serves as a sulfhydryl buffer that maintains the cysteine residues of hemoglobin and other proteins in a reduced state. Glutathione also plays a key role in detoxifying the body by reacting with both endogenous and exogenous compounds, such as hydrogen peroxide and other peroxides.
Evidence suggests that glutathione is useful at protecting the body from the harmful side effects of radiation and chemotherapy that often accompany cancer treatment. Cyclophosphamide (CTX), for example, is a widely used antitumor agent whose clinical utility is limited by its bladder toxicity. During CTX metabolism in the body, a compound, acrolein, is released. Acrolein is thought to be responsible for the urotoxicity of CTX. Glutathione has been implicated in CTX detoxification by conjugating to acrolein.
It has been of significant interest in the art, therefore, to increase glutathione synthesis especially during periods of toxic insults. L-cysteine, a reactant in normal glutathione biosynthesis, is known to increase the synthesis of endogenous glutathione. To date, a significant challenge in the art has been to provide L-cysteine to cells at sufficiently high levels to drive glutathione biosynthesis. As disclosed, for example, in U.S. Pat. No. 4,868,114 to Nagasawa et al., prodrugs of L-cysteine (i.e., chemical compounds converted to L-cysteine in the cell), such as RibCys, can be used by the cell to drive glutathione biosynthesis shown below. 
These prodrugs have been shown to offer good protection against a variety of toxic insults. However, the initial prodrugs are highly water soluble and are rapidly excreted by the body.
WR compounds are also of significant interest in the art. Over 4400 WR compounds were prepared and tested at the Walter Reed Army Hospital after World War II in an effort to develop radioprotective compounds that might be employed by military personnel during a nuclear encounter. The single agent with the greatest potential that arose from that extensive effort was WR-2721, which is converted to WR-1065 by enzymatic cleavage. These compounds have several shortcomings, however, including that they possess noteworthy toxicity and little oral activity, greatly reducing their clinical utility.
Finally, selenocysteines are of significant interest in the art for their antioxidant and anticancer properties. In fact, selenium has received significant attention for its ability to inhibit or delay the onset of AIDS caused by HIV infection. Selenium is also a cofactor of glutathione peroxidase, an enzyme which has been implicated in many detoxifying processes.
Selenium is an essential mineral that is critical to the normal functioning of many species, including humans. It also has demonstrated activity as a cancer chemopreventive agent. Selenium-containing compounds appear to have especially high preventive activity against the initiation phase of colorectal cancer, although its chemoprotective ability has been extended to cancers in many organs, caused by a variety of carcinogens.
To achieve this chemopreventive activity, levels of selenium at least five-fold greater than that required for normal nutritional status appear to be necessary. In addition, selenium must be given continuously for maximum inhibition. Unfortunately, selenium is also known for its profound toxicity, making selenium supplementation a distinct challenge.
Current selenium supplements rely on inorganic forms, such as sodium selenite (Na2SeO3) or sodium selenate (Na2SeO4). While these forms have some value, they are considered more toxic than necessary, and are unlikely to be useful in cancer chemo-prevention. Several organoselenium compounds, which appear to be less toxic in general than the inorganic forms, have been proposed for in vivo use, but the full potential of this strategy has not yet been realized. In general, however, it is very clear that the chemical form in which selenium is introduced consistently shows a marked influence on biological outcomes, including cancer chemoprevention and toxicity.
Selenocysteine is an organic form that is present in the body and is now recognized as the 21st amino acid used in protein synthesis. While it represents a valuable biochemical form, selenocysteine is chemically unstable and difficult to handle, which has undoubtedly deterred its study and use. In addition, even though it possesses greatly reduced inherent toxicity, it still may be too toxic at chemopreventive doses to the therapeutically useful. Accordingly, prodrug forms of selenocysteine that possess reduced inherent toxicity and improved physicochemical properties would be desirable.
Objects of the Invention
It is, therefore, an object of the invention to provide prodrugs and conjugates of thiol- and selenol-containing compounds, such as cysteine, cysteamine, glutathione, selenocysteine, selenocysteamine, and the WR compounds.
Another object of the invention is to provide such thiol- or selenol-containing compounds displaying reduced toxicity and increased clinical utility.
Another object of the invention is to provide such thiol- or selenol-containing compounds with increased lipophilicity that can target a specific organ or region of the body.
Another object of the invention is to provide such thiol- or selenol-containing compounds that can be conjugated to antioxidants, such as vitamin C and E, thus maximizing the effects by providing different agents that work by complementary mechanisms.
Further objects of the invention will become evident in the description below.
The present invention is directed to novel prodrugs and conjugates of thiol- or selenol-containing compounds, including cysteine, cysteamine, glutathione, selenocysteine, selenocysteamine, and the WR compounds. Potential protective or preventive uses of such agents are widespread, as in reducing the unwanted side effects of chemo- or radiotherapy of cancer, improving cardiovascular function, preventing mutagenesis, preventing the initiation and/or progression of cancer, reducing toxic consequences of planned or unplanned radiation or chemical exposures, slowing the aging process, preventing cataract formation, etc.
Prodrugs are inactive forms of a parent drug that have been created to overcome one or more barriers to their effective use. In the present invention, prodrugs have been designed to overcome the chemical instability and/or possible toxicity barriers that exist with the parent drug.
In one embodiment, the invention relates to the design, synthesis, and evaluation of prodrugs of L-cysteine and L-selenocysteine, containing a thioglycoside or selenoglycoside on the free thiol or selenol. The protecting group will, in addition to protecting the thiol or selenol from oxidation, permit the targeting of specific sites within the body.
For example, the galactose protected cysteine shown below will target the liver and will enter the cytoplasm of hepatocytes. Delivering L-cysteine to hepatocytes has numerous uses, including protection against hepatotoxins, such as acetaminophen, and against side effects caused by local radiation treatments. 
The cysteine/selenocysteine prodrugs can be depicted by the formula: 
where A is a sulfur or a selenium, and R is derived from a mono- di- or oligo-saccharide, such as ribose, galactoxe, glucose, or mannose.
A second embodiment relates to the design, synthesis, and evaluation of novel prodrugs that are derivatives of cysteamines or selenocysteamines, such as of WR compounds, particularly WR-1065. The prodrug strategy is similar to that employed for L-cysteine, using a protecting group Rxe2x80x2. R is typically a sugar, such as ribose. The modified WR prodrugs have numerous uses including protection against the side effects of radiation and chemotherapy, radiation and chemical induced mutations, such as from exposure to radiation during a nuclear accident or chemical spill, and even spontaneous mutations which are the cause of most cancers.
These prodrugs can be described by the formulas; 
where A is sulfur or selenium, Rxe2x80x2 is derived from a sugar and has the formula (CHOH)nCH2OH, where n is 1 to 5. Rxe2x80x2 may also be hydrogen, an alkyl or aryl group, such as methyl, ethyl, benzyl, carboxyl, polyhydroxyalkyl, or phenyl, or may also be xe2x95x90O. The Rxe2x80x3 groups may be the same or different and may be alkyl, alkoxy, carboxy, such as acetyl, methyl or ethyl.
These novel thio- and selenol-containing compounds overcome several problems facing the art, including toxicity, water-solubility, and lack of target specificity. First, the protective or preventive activity and clinical utility will be greatly enhanced by converting the cysteine, cysteamine, glutathione, selenocysteine, selenocysteamine, and WR compounds, to thiazolidine and selenazolidone prodrug forms. These prodrugs provide a slow release form of the thiol-/selenol-amine, which greatly reduces observed toxicity (with related compounds), but provides the active agent after enzymatic or non-enzymatic biotransformation
In a third embodiment, the invention relates to the design, synthesis, and evaluation of novel covalent conjugates of thiolamines or selenolamines and antioxidant vitamins, e.g., Vitamin E and Vitamin C. These compounds include conjugates of any of the prodrug compounds of the invention defined above conjugated with Vitamin C or Vitamin E. Also contemplated by the invention are conjugates of antioxidant vitamins with the following thiol- and selenol-amines and derivatives thereof; cysteine, cystine, cysteamine, cystamine, glutathione, selenocysteine, selenocysteamine, selenocystine, selenocystamine, and WR compounds (WR-1065 and WR-33278).
An example, shown below is a conjugate of cysteamine and Vitamin C. 
These compounds are effective because protective or preventive treatment of toxic insult will be far more effective if thiol- or selenol-containing compounds are delivered together with antioxidants such as vitamin C and E which also play a protective and preventative action in the body. The complementary mechanisms of these compounds would increase the overall effectiveness of treatment.
In yet another embodiment, the invention relates to the design, synthesis, and evaluation of novel L-cysteine prodrugs which have been modified with ester or amine groups at the carboxylic acid position. These can be described by formula; 
where A is sulfur or selenium, and Rxe2x80x2 is derived from a sugar and has the formula (CHOH)nCH2OH, where n is 1 to 5. Rxe2x80x2 may also be an alkyl or aryl group, such as methyl, ethyl, benzyl, carboxyl, or phenyl, or may also be xe2x95x90O. R‡ is an alkoxy, such as xe2x80x94OR1 where R1 is ethyl, methyl, R‡ may also be an amine group (xe2x80x94NRxe2x80xa02) where the Rxe2x80xa0 groups are the same or different and hydrogen or an alkyl group, such as methyl.
Yet another embodiment of the invention is the condensation product of a selenolamine and a carbonyl donor characterized by the formula: 
where R is COOH (prodrug of L-selenocysteine) of is H (prodrug of selenocysteamine). Rxe2x80x2 is derived from a sugar and has the structure (CHOH)nCH2OH. and where n is 1 to 5; an alkyl or aryl group, such as methyl, ethyl, benzyl, phenyl or carboxyl; or xe2x95x90O.